盐生草属染色体核型分析及盐生草相关耐盐基因的克隆和遗传转化

发布时间：2018-01-26 01:36

本文关键词： 盐生草属核型分析耐盐基因克隆植物表达载体　出处：《甘肃农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：盐胁迫是影响植物生长发育,造成作物产量降低的主要非生物因素之一。它对植物的危害主要表现在渗透压胁迫和离子胁迫等方面,使植物体细胞膜结构、酶活性、光合作用、呼吸作用器官受到影响,且在植物体内作用产生活性氧,导致氧化胁迫,使农作物生长失衡,产量降低甚至死亡。改良盐碱地可通过改良土壤、合理灌溉等农业措施,或利用基因工程技术培育耐盐碱作物品种以提高作物的适应性。盐生草和白茎盐生草都属于藜科盐生草属一年生草本植物,分布于我国的甘肃西北部、新疆地区、青海地区及西藏地区等,是一种典型的盐生植物。对这两种植物的染色体核型进行分析,为开展盐生草基因组测序的准确分析及我国盐生植物的种类鉴别、亲缘关系远近和种质资源的遗传多样性提供细胞学依据。盐生草植物地上有很多分枝的茎,具有保持水土、防止风沙、耐盐和抗旱能力,其自身蕴含了很多的耐盐基因,尤其在植物对盐碱混合胁迫下基因表达差异性的表现,极有可能挖掘大量与耐盐碱方面的有利基因,对改良作物性状及培育耐盐碱作物具有重要意义。下列内容为本实验的研究结果:1.盐生草属染色体核型分析通过对盐生草属的两种盐生植物进行核型分析,其结果为:盐生草和白茎盐生草植物染色体数目均为18条,二倍体植物,染色体基数均为9,核型公式分别表示为2n=2x=18=12m+6sm和2n=2x=18=16m+2sm,都是由近中部着丝点区(sm)和中部着丝点区(m)构成。相对长度分别在7.808%-14.888%和7.50%-15.13%之间。核不对称系数分别为61.908%和41.662%,其核型分别为“2A”和“1B”型,由结果可以看出白茎盐生草属于较原始的物种。2.相关耐盐基因的克隆①以盐生草cDNA为模板,利用RACE技术与RT-PCR方法获得了Unigene19352和Unigene18968的3’端序列,经过拼接得到3’端全长序列分别为555 bp和608 bp,为后期cDNA全长的获得奠定了基础。②采用RT-PCR方法从盐生草中克隆到Unigene19095的cDNA全长序列。分析结果为:此基因cDNA全长为678 bp,包含3’UTR区388个核苷酸,5’UTR区29个核苷酸,开放阅读框长度261 bp,编码86个氨基酸,分子量为9.67 kDa。软件预测该基因编码的蛋白存在信号肽,含有两个跨膜区域,二级结构中α螺旋,延伸链,随机卷曲,β-转角各占22.09%,36.05%,37.21%、4.65%,含有3个磷酸化位点,但该基因的具体功能需要更深层次研究确定。3.植物表达载体CPB-Unigene19095的构建及转化成功构建表达载体CPB-Unigene19095,并转入农杆菌LBA4404,采用蘸花法侵染拟南芥花序,获得拟南芥抗性植株T1代。经过PCR检测,初步确定Unigene19095已成功转入拟南芥中,为进一步得到纯合的转基因拟南芥植株以及其后代的表型检测,功能验证奠定了基础。
[Abstract]:Salt stress is one of the main abiotic factors that affect the growth and development of plants and reduce crop yield. Its harm to plants is mainly reflected in osmotic stress and ion stress. Enzyme activity, photosynthesis and respiration are affected, and reactive oxygen species (Ros) are produced in plants, which lead to oxidative stress and make crops grow out of balance. The improvement of saline-alkali land can be done by improving soil, reasonable irrigation and other agricultural measures. In order to improve the adaptability of saline-tolerant crops, both halophyte and salt-stem halophytes belong to the annual herbaceous plants of the genus Chenopodiaceae, which are distributed in the northwest of Gansu Province. Xinjiang, Qinghai and Tibet are typical halophytes. For the accurate analysis of the genome sequencing of halophytes and the species identification of halophytes in China, the genetic diversity of germplasm resources and the genetic diversity of the genetic resources of halophytes were provided with cytological basis. There were many branched stems on the ground of halophytes. With the ability of soil and water conservation, sand prevention, salt tolerance and drought resistance, it contains a lot of salt tolerance genes, especially the difference of gene expression under saline-alkali mixed stress. It is highly likely to dig up a large number of favorable genes related to salt and alkali tolerance. It is of great significance to improve crop characters and cultivate saline-tolerant crops. The following contents are the results of this study: 1. Karyotype analysis of two halophytes of halophytes is carried out by means of karyotype analysis of halophytes. The results were as follows: the number of chromosomes of halophytes and albicans was 18, and the chromosome number of diploid plants was 9. The karyotype formula is expressed as 2ng ~ 2x ~ (18) 12m ~ 6sm and 2n ~ 2x ~ (18) ~ (18) ~ (16) m ~ (2sm) respectively. Both from the near central metacentric area (Sm) and from the central centromere region (Mm). The relative length was between 7.808% -14.88% and 7.50-15.13%. The nuclear asymmetry coefficient was 61.908% and 41.662%, respectively. The karyotypes were "2A" and "1B", respectively. From the results, it can be seen that the halophytes belong to the more primitive species. 2. Clone 1 of the related salt tolerance gene takes cDNA as the template. The 3'end sequences of Unigene19352 and Unigene18968 were obtained by using RACE technique and RT-PCR method. The full-length sequence of 3 'end was 555 BP and 608 BP, respectively. 2. The whole cDNA sequence of Unigene19095 was cloned from halophytic grass by RT-PCR method. The results were as follows:. The cDNA length of the gene is 678bp. There are 388 nucleotides in UTR region and 29 nucleotides in 5 UTR region. The open reading frame is 261 BP long and encodes 86 amino acids. The molecular weight of the protein was 9.67 kDa.The software predicted that the protein encoded by the gene contained two transmembrane regions, a helix in the secondary structure, extended chains and random curls. The 尾 -turning angle accounted for 22.09% and 36.05%, and 37.21%, 4.65%, containing three phosphorylated sites. However, the specific function of the gene needs to be further studied and determined. 3. The construction and transformation of plant expression vector CPB-Unigene19095 and the successful construction of expression vector CPB-Unigene1. 9095. And transferred to Agrobacterium tumefaciens LBA4404, infected Arabidopsis thaliana inflorescence with the method of dipping flowers, and obtained T1 generation of Arabidopsis resistant plants. The results were detected by PCR. It was preliminarily confirmed that Unigene19095 had been successfully transferred into Arabidopsis thaliana, which laid a foundation for the phenotypic detection and functional verification of homozygous transgenic Arabidopsis thaliana plants and their progenies.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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